Understanding the processes that lead from a fertilized egg to the formation of germ layers and subsequently to a body plan is a central goal of embryology. Much of what is known about the development of a vertebrate body plan comes from studies of amphibia where, at the tadpole stage, the main body axis consists of the dorsal structures notochord, spinal cord and somites organized anterior to posterior as head, trunk and tail. All animal tissues derive from the three germ layers and the mesoderm plays a pivotal role in organizing the body axis (Keller, R. in Methods in Cell Biology, eds Kay and Peng, Academic Press: San Diego, 1991). Mesodermal cells lead the movements of gastrulation (Keller et al. (1988) Development 103:193-210; and Wilson et al. (1989) Development 105:155-166), are required for the patterning of the nervous system (Mangold et al. (1933) Natyrwissenschaften 21:761-766; and Hemmati-Brivanlou et al. (1990) Science 250:800-802), and themselves give rise to the muscular, skeletal, circulatory and excretory systems. Moreover, a portion of the dorsal mesoderm from early gastrula, the Spemann organizer, can induce and organize a second body axis following transplantation to another site (Spemann et al. (1924) Arch mikr Anat EntwMech 100:599-638).
The origin of the nervous system in all vertebrates can be traced to the end of gastrulation. At this time, the ectoderm in the dorsal side of the embryo changes its fate from epidermal to neural. The newly formed neuroectoderm thickens to form a flattened structure called the neural plate which is characterized, in some vertebrates, by a central groove (neural groove) and thickened lateral edges (neural folds). At its early stages of differentiation, the neural plate already exhibits signs of regional differentiation along its anterior posterior (A-P) and mediolateral axis (M-L). The neural folds eventually fuse at the dorsal midline to form the neural tube which will differentiate into brain at its anterior end and spinal cord at its posterior end. Closure of the neural tube creates dorsal/ventral differences by virtue of previous mediolateral differentiation. Thus, at the end of neurulation, the neural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see, for example, Principles in Neural Science (3rd), eds. Kandel, Schwartz and Jessell, Elsevier Science Publishing Company: NY, 1991; and Developmental Biology (3rd), ed. S. F. Gilbert, Sinauer Associates: Sunderland Mass., 1991).
Before gastrulation the three germ layers are simply arranged, top to bottom, in a frog blastula. Ectoderm arises from the top, or animal pole; mesoderm from the middle, or marginal zone, and endoderm from the bottom or vegetal pole. Mesoderm can be induced in animal pole cells (animal caps) by signals emanating from the vegetal pole. Several peptide growth factors have been identified that can induce mesoderm in animal caps in vitro. When animal cap tissue is explanted from a blastula embryo and cultured in isolation it develops into a ball of epidermis. But in the presence of a mesoderm inducing factor, the animal cap will differentiate into mesodermal derivatives, including notochord, muscle and blood. Members of the fibroblast growth factor family, in particular basic fibroblast growth factor (bFGF), and the transforming growth factor-xcex2 (TGF-xcex2) family, notably activins and Vg-1, are potent inducers in this assay. Xenopus homologues of the Wnt gene family may also have a role in mesoderm induction. Both Xwnt1 (McMahon et al. (1989) Cell 58, 1075-1084) and Xwnt8 messenger RNAs elicit dorsal mesoderm formation when injected into the ventral side of an early embryo, an activity shared by Vg-1, and to a lesser extent by activin RNA. bFGF and activin protein can be detected in the early embryo and although there are no data on the localization of activin, there is evidence that bFGF is present in the marginal zone and vegetal pole of early blastula. Vg-1 is present at the appropriate time and in the right region known to be responsible for mesoderm induction in vivo. Although Xwnt1 and Xwnt8 are not present at the proper time or place to effect dorsal mesoderm induction, there may be other Xwnts that fulfill this role.
Many types of communication take place among animal cells. These vary from long-range effects, such as those of rather stable hormones circulating in the blood and acting on any cells in the body that possess the appropriate receptors, however distant they are, to the fleeting effects of very unstable neurotransmitters operating over distances of only a few microns. Of particular importance in development is the class of cell interactions called embryonic induction; this includes influences operating between adjacent cells or in some cases over greater than 10 cell diameters (Saxen et al. (1989) Int J Dev Biol 33:21-48; and Gurdon et al. (1987) Development 99:285-306). Embryonic induction is defined as in interaction between one (inducing) and another (responding) tissue or cell, as a result of which the responding cells undergo a change in the direction of differentiation. This interaction is often considered one of the most important mechanism in vertebrate development leading to differences between cells and to the organization of cells into tissues and organs. Adult organs in vertebrates, and probably in invertebrates, are formed through an interaction between epithelial and mesenchymal cells, that is, between ectoderm/endoderm and mesoderm, respectively.
The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another, by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
While there has been considerable progress in identifying molecules responsible for mesoderm induction, practically nothing is known about the molecular nature of neural induction. Candidate neural patterners are growth factors that are involved in mesoderm patterning in earlier stages and become localized later in a subset of cells in the nervous system. These molecules include different members of the Wnt, TGF-xcex2 and FGF families. Three members of the Wnt family Wnt-1, Wnt-3 and Wnt-3A, are localized in the roof plate (dorsal spinal cord) and a subset of brain cells. Good evidence that Wnt products pattern the neural tube comes from homozygote mice lacking the Wnt-1 gene product; these mutant mice display a strong abnormality in the anterior hindbrain and posterior midbrain (a region that coincides with engrailed-2 expressing cells)(McMahon et al. (1992) Cell. 69:581-595). Vg-1, BMP-4 (Jones et al. (1991) Development. 111:532-542) and dorsalin-1 (Blumberg et al. (1991) Science 253:194-196) are examples or TGF-xcex2 family members that display restricted expression in the embryonic nervous system (see also, Lyons et al. (1991) Trends Genet 7:408-412; and Massague et al. (1990) J Biol Chem 265:21393-21396). Dorsalin-1 inhibits the differentiation of motor neurons and induces migration of neural crest cells and thus may be involved in dorsal ventral patterning of the neural tube (Blumberg et al. (1991) Science 253:194-196). Finally acidic FGF (aFGF), basic FGF (bFGF) as well as the newly characterized FGF from Xenopus embryos, XeFGF, (Isaacs et al. (1992) Development. 114:711-20) are all expressed in some cells of the developing neural tube (Weise et al. (1992) Cell and Tissue Research. 276:125-130; and Tannahill et al. (1992) Development. 115:695-702).
Since the natural embryonic neural inducer or patterner has yet to be characterized, the analysis of the mechanisms of induction and patterning is difficult. However, studies have demonstrated that notochord can induce and pattern neural structures (Jones et al. (1989) Development. 107:785-791; and Sharpe et al. (1987) Cell. 50:749-758) which implies that the signals can travel vertically from the axial mesoderm to the overlying ectoderm. The finding that neuralization can be induced by mesoderm suggests that neural induction involves a signal acting in a paracrine fashion, the transduction of which appears to involve protein kinase C (Otte et al. (1991) Science. 251:570-573). A recent series of experiments, exploring one of Spemann""s original ideas, have demonstrated that signals involved in both induction and patterning of the nervous system can also travel through the plane of the ectoderm (Dixon et al. (1989) Development 106:749-757; Doniach et al. (1992) Science 257:542-545; and Ruiz i Altaba, A. (1992) Development. 115:67-80). It is now accepted that both types of mechanisms coexist in the embryo and play a role in neurogenesis.
The present invention makes available a method for inducing neuronal differentiation and preventing the death and/or degeneration of neuronal cells both in vitro and in vivo. The subject method stems from the unexpected finding that, contrary to traditional understanding of neural induction, the default fate of ectodermal tissue is neuronal rather than mesodermal and/or epidermal. In particular, it has been discovered that preventing or antagonizing a signaling pathway in a cell for a growth factor of the TGF-xcex2 family (hereinafter xe2x80x9cTGF-xcex2-type growth factorxe2x80x9d), can result in neuronal differentiation of that cell. In the subject method, signaling by the TGF-xcex2-type growth factor is disrupted by antagonizing the inhibitory activity of the TGF-xcex2-type growth factor. For instance, this can be accomplished by sequestering the growth factor with a growth factor binding protein (such as an activin-binding protein where the neural-inhibitory growth factor is activin) or by treating with an antagonist which competes with the growth factor for binding to a growth factor receptor on the surface of the cell of interest.
In one embodiment of the subject method, inducing cells to differentiate to a neuronal cell phenotype comprises contacting the cells with an agent which antagonizes the biological action of at least one polypeptide growth factor of the TGF-xcex2 family which normally induces the cells to differentiate to a non-neuronal phenotype. The antagonizing agent can inhibit the biological activity of the TGF-xcex2-type growth factor, for example, by preventing the growth factor from binding its receptors on the surface of the treated cells. In another embodiment, the antagonizing agent binds to the growth factor and sequesters the growth factor such that it cannot bind its receptors.
To further illustrate the invention, the antagonizing agent can be selected from a group consisting of a follistatin, an xcex12-macroglobulin, a protein containing at least one follistatin module, and a truncated receptor for a growth factor of the TGF-xcex2 family. In the instance of the truncated receptor, it can be a soluble growth factor-binding domain of a TGF-xcex2 receptor, or, in another embodiment, the truncated receptor can be a membrane bound receptor and comprises an extracellular growth factor-binding domain of a TGF-xcex2 receptor, a transmembrane domain for anchoring the extracellular domain to a cell surface membrane, and a dysfunctional cytoplasmic domain. In the latter embodiment, the truncated receptor is recombinantly expressed in the treated cell.
In certain embodiments of the present method, the TGF-xcex2-type growth factor which inhibits neuronal differentiation is an activin. In such instances, the method comprises contacting the cells with an agent which disrupts the activin signaling pathway in the cells, causing the cells to default to neuronal differentiation, rather than, for instance, mesodermal and/or epidermal fates.
The present method can be used in vitro, for example, to induce cells in culture to differentiate to a neuronal phenotype. Moreover, the present method is amenable to therapeutic application, and as described below, can be used to treat neurodegenerative disorders associated with, for example, the progressive and persistent loss of neuronal cells, such as which occurs with Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, Pick""s disease, Huntington""s disease, multiple sclerosis, neuronal damage resulting from anoxia-ischemia, neuronal damage resulting from trauma, and neuronal degeneration associated with a natural aging process.
As described herein, a collective group of experiments performed with either a truncated activin receptor conferring a dominant negative effect, or with a recombinant follistatin or inhibin, establish that a signaling pathway of a growth factor of the TGF-xcex2 family is involved in inhibiting neural induction in vivo. The present findings indicate, for the first time in vertebrates, that neuralization is a default state. As described in the Examples below, our results indicate that activin, or any other member of the TGF-xcex2 family that interacts with the truncated activin receptor, can inhibit neural induction, as these TGF-xcex2 signals instruct cells towards non-neuronal facts such as epidermal, mesodermal or endodermal fate. Inhibition of signal transduction by a TGF-xcex2-type growth factor, by either the truncated activin receptor, follistatin, or inhibin, induced cells of the intact animal cap to switch to a neuronal fate in the absence of any detectable mesoderm. This data indicates that presumptive neural tissue in animal caps can respond to TGF-xcex2-type growth factor and form mesodermal and/or epidermal tissues, but if this tissue specification by the factor is blocked, the cells become neural. As described below, activin is strongly implicated as the TGF-xcex2-type growth factor that inhibits neuronal differentiation. Both activin and its receptor are present naturally in the animal cap, indicating that an endogenous activity that blocks activin signaling switches the tissue from an ectodermal to a neural fate. Thus, endogenous activin may act as a neural inhibitor (acting to induce epidermal development and/or mesoderm), and neuralization requires the inhibition of activin activity.
The present invention makes available a method for inducing neuronal differentiation and/or preventing the death or degeneration of neuronal cells. In general, the method comprises contacting a cell, either in vivo or in vitro, with an agent capable of antagonizing the biological action of a protein from the family of transforming growth factor-xcex2s. The mechanism of action of the antagonist can, for example, comprise: competitive or non-competitive binding to a cell-surface receptor for the growth factor; sequestration of the growth factor; or inhibition of signal transduction events mediated by the growth factor receptor. Representative embodiments are described in more detail below.
The subject method stems from the unexpected finding that, contrary to traditional understanding of neural induction, the default fate of ectodermal tissue is neuronal rather than epidermal. In particular, it has been discovered that preventing or antagonizing a TGF-xcex2-type growth factor signaling pathway for a cell can result in neuronal differentiation of that cell. In the subject method, signaling by the TGF-xcex2-type growth factor is disrupted by antagonizing the inhibitory activity of the TGF-xcex2-type growth factor. For instance, this can be accomplished by sequestering the growth factor with a growth factor binding protein (such as an activin-binding protein) or by treating with an antagonist which competes with the growth factor for binding to a growth factor receptor on the surface of the cell of interest.
As described herein, the present method can be used in vitro, for example, to induce cells in culture to differentiate to a neuronal phenotype. In one embodiment, the differentiated cells are subsequently continued in culture, and can be used to provide useful in vitro assay systems as well as valuable research tools for further understanding neural development. In another embodiment, the differentiated cells are used in vivo for transplantation. Moreover, the present method is amenable to therapeutic application, and as described below, can be used to treat neurodegenerative disorders associated with, for example, the progressive and persistent loss of neuronal cells, such as which occurs with Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, and Huntington""s disease.
While the following description, for clarity, describes the use of agents which antagonize activin signaling, it is understood that many such agents can also bind or otherwise antagonize other TGF-xcex2-type growth factors and thereby disrupt their inhibition, if any, of neuralization. As used herein, the terms xe2x80x9ctransforming growth factor-betaxe2x80x9d and xe2x80x9cTGF-xcex2xe2x80x9d denote a family of structurally related paracrine polypeptides found ubiquitously in vertebrates, and prototypic of a large family of metazoan growth, differentiation, and morphogenesis factors (see, for review, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; and Sporn et al. (1992) J Cell Biol 119:1017-1021). Moreover, the present invention, namely the discovery that neuralization is a default state, will readily allow identification of other factors, including other TGF-xcex2-like growth factors, which inhibit a cell from reaching this default (e.g. actively induce non-neuronal differentiation). In light of this understanding, agents which disrupt these factors are specifically contemplated by the present invention.
An agent capable of antagonizing the signaling pathway of a TGF-xcex2 factor involved in preventing neuronal differentiation, so as to cause a cell to default to neuronal differentiation, is herein referred to as a neuralizing agent, or xe2x80x9cNAxe2x80x9d.
In one embodiment, the NA is an activin-binding protein which can reduce the bioavailability of activin, e.g. by sequestering activin in the extracellular milleu, with exemplary activin-binding agents including follistatins, xcex12-macroglobulin, and activin receptors. Other activin-binding proteins of the present method can include agrin, agrin-related proteins, and other proteins containing follistatin modules. In a preferred embodiment, the NA has a binding affinity for activin on the order of, or greater than, that of either a follistatin or an activin receptor.
In an illustrative embodiment of the present method, the NA is a follistatin able to bind and sequester activin. Follistatins are single chain, glycosylated polypeptides that were first isolated based on their ability to inhibit follicle-stimulating hormone release. Follistatins from several species, including human, have been structurally characterized and cloned. (See, for example, Esch et al. (1987) Mol. Endocrinology 11:849; Ling et al. International Publication No. WO 89/01945; Ling et al. U.S. Pat. No. 5,182,375; Ling et al. U.S. Pat. No. 5,041,538; and Inouye et al. (1991) Endocrinology 129:815-822). For instance, two forms of human follistatin have been cloned and expressed, one having 315 amino acid residues, and one having 288 amino acid residues. (Inouye et al., supra). Human follistatin is available through the National Hormone and Pituitary Program of the NIH. In a one embodiment, the follistatin of the present method is of the class of shorter follistatins (e.g. the 288 a.a. human homolog), since, as described below, these forms appear to have a greater binding affinity for activin, relative to the larger forms of follistatin.
In another exemplary embodiment, the activin-binding protein can be an activin receptor, or portion thereof. Activin receptors have also been cloned from several species. (Attisano et al. (1992) Cell 68:97-108; and Gerogi et al. (1990) Cell 61:635-645). In embodiments of the present invention in which it is desirable for the NA to be a diffusible molecule, a soluble extracellular portion of an activin receptor can be used, provided the extracellular portion is chosen so as to retain activin-binding. For example, a soluble form of an activin receptor can be generated using the cloned activin receptor gene of Attisano et al., which includes an endogenous signal sequence for secretion (Attisano et al. (1992) Cell 68:97-108). For instance, a stop codon can be introduced at a site 5xe2x80x2 of the gene encoding the transmembrane domain (e.g. the ACC encoding Thr-134 can be mutated to TAA). Moreover, as described below, the truncated activin receptor can be engineered as fusion protein to include other polypeptide sequences.
In yet another illustrative embodiment of the present invention, the cell can be contacted with an activin antagonist which inhibits activin binding to its cognate receptor on the treated cell by competitively, or non-competitively, binding to the receptor protein. Such neuralizing agents can be utilized to block activin signaling and thereby induce the treated cell to undergo neuronal differentiation or to maintain its existing neuronal differentiation. A number of potential activin antagonists of this type are known in the art, including the family of inhibins. Inhibins and activins were first isolated and purified from follicular fluid on the basis of their ability to inhibit (inhibin) or stimulate (activin) FSH release by pituitary cells. Mature inhibin is typically a heterodimeric glycoprotein composed of a common xcex1-subunit and one of two xcex2 subunits, xcex2A and xcex2B. In addition to inhibins, the subject invention can be carried out using activins that have been mutagenized to create activin variants which act antagonistically to activin in neuronal induction. Activins are homodimeric forms of inhibin xcex2-subunits (e.g. xcex2Axcex2A or xcex2Axcex2B). Such antagonists can be generated, for example, by combinatorial mutagenesis techniques well known in the art (See, for example, Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461). Furthermore, peptidomimetics (e.g. of activin or inhibin) or other small molecules, such as may be identified in the assays set out below, can be used to antagonize activin signalling by binding to the receptor and precluding functional binding (or receptor oligomerization) by activin.
In still further embodiments, the neuralizing agent acts to block signal transduction by the activin receptor irrespective of activin binding. Such agents include dominant negative receptors which, unlike the soluble form of the receptor, are membrane bound, e.g. which include a transmembrane domain and at least a portion of a cytoplasmic domain. Such receptors, rather than merely sequestering activin from functional receptors, are incapable of activating appropriate intracellular second messenger pathways in response to activin binding. Expression of these dominant negative receptors in cells expressing wild-type receptor can render the cells substantially insensitive to activin, e.g. by formation of non-productive oligomers with the wild-type receptor. Likewise, other agents which inhibit the activin receptor second messenger pathways downstream of the activin receptor can be used to inhibit activin-mediated induction of cells.
Yet another embodiment of the subject assay features the use of an isolated nucleic acid construct for inhibiting synthesis of an activin receptor in the targeted cell by xe2x80x9cantisensexe2x80x9d therapy. As used herein, xe2x80x9cantisensexe2x80x9d therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridizes (e.g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an activin receptor so as to inhibit expression of that receptor, e.g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, xe2x80x9cantisensexe2x80x9d therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an activin receptor. Alternatively, the antisense construct can be an oligonucleotide probe which is generated ex vivo and which, when introduced into the treated cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an activin receptor gene. Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.
In certain embodiments, when appropriate, the neuralizing agent can be a chimeric protein comprising a moiety that binds a component of the extracellular matrix. Such a chimeric NA can be useful in circumstances wherein diffusion of the NA from a treatment site is undesirable, and will function to such an end by virtue of localizing the chimeric NA at or proximate a treatment site. An NA of this embodiment can be generated as the product of a fusion gene, or by chemical cross-linking. For instance, a number of proteins have been characterized from the extracellular matrix (ECM) of tissues that will support the localization of a chimeric NA at a target site. One example of a well characterized protein is fibronectin. Fibronectin is a large adhesive glycoprotein with multiple functional domains. Several of these domains have matrix attachment activity. For example, one of these is a single xe2x80x9ctype-III repeatxe2x80x9d which contains a tetrapeptide sequence R-G-D-S (Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al. (1985) EMBO 4:1755-9). Peptides as small as pentapeptides containing these amino acids are able to support attachment to a cell through binding ECM components (Ruoslahti et al. (1987) Science 238:491-497; Pierschbacher et al. (1987) J. Biol. Chem. 262:17294-8.; Hynes (1987) Cell 48:549-54; and Hynes (1992) Cell 69:11-25). In fact, several companies have commercialized products based on this cell attachment sequence for use as reagents in cell culture and various biomaterials applications. See for example recent catalogs from Telios Pharmaceutical, BRL, Stratagene, Protein Polymer Technologies etc., as well as U.S. Pat. Nos. 4,517,686; 4,589,881; 4,578,079; 4,614,517; 4,661,111; and 4,792,525. Accordingly, fibronectin binding sequences can be added, for example, to the soluble activin receptor described herein.
Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, and/or enhancing survival of a neural cell responsive to activin induction, by contacting the cell with a neuralizing agent (e.g. an activin antagonist). For instance, it is contemplated by the invention that, in light of the present finding of an apparently broad involvement of activin in the formation of ordered spatial arrangements of differentiated neural tissues in vertebrates, the subject method could be used to generate and/or maintain an array of different neural tissue both in vitro and in vivo. The neuralizing agent can be, as appropriate, any of the preparations described above, including isolated polypeptides, gene therapy constructs, antisense molecules, peptidomimetics or agents identified in the drug assays provided herein.
For example, the present method is applicable to cell culture technique. In vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). Once a neuronal cell has become terminally-differentiated it typically will not change to another terminally differentiated cell-type. However, neuronal cells can nevertheless readily lose their differentiated state. This is commonly observed when they are grown in culture from adult tissue, and when they form a blastema during regeneration. The present method provides a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of other trophic factors. In such embodiments of the subject method, the cultured cells can be contacted with a neuralizing agent of the present invention in order to induce neuronal differentiation (e.g. of a stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of differentiation. The source of the neuralizing agent in the culture can be derived from, for example, a purified or semi-purified protein composition added directly to the cell culture media, or alternatively, released from a polymeric device which supports the growth of various neuronal cells and which has been doped with the neuralizing agent. If appropriate, the source of the neuralizing agent can also be a cell that is co-cultured with the intended neuronal cell and which produces a recombinant neuralizing agent. Alternatively, the source can be the neuronal cell itself which as been engineered to produce a recombinant neuralizing agent. In an exemplary embodiment, a naive neuronal cell (e.g. a stem cell) is treated with an activin antagonist in order to induce differentiation of the cells into, for example, sensory neurons or, alternatively, motorneurons. Such neuronal cultures can be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments.
To further illustrate potential uses, it is noted that intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate behavioral defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells. Thus, use of activin antagonist for maintenance of neuronal cell cultures can help to provide a source of implantable neuronal tissue. The use of a neuralizing agent in the culture can be to prevent loss of differentiation, or where fetal tissue is used, especially neuronal stem cells, a neuralizing agent of the present invention can be used to induce differentiation.
Stem cells useful in the present invention are generally known. For example, several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which can generate only one type of cell, such as sensory neurons, and likely represent committed progenitor cells. The role of an activin-disrupting agent employed in the present method to culture such stem cells can be to induce differentiation of the uncommitted progenitor and thereby give rise to a committed progenitor cell, or to cause further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally-differentiated neuronal cell. For example, the present method can be used in vitro to induce and/or maintain the differentiation of neural crest cells into glial cells, schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, as well as peptidergic and serotonergic neurons. The neuralizing agent can be used alone, or can be used in combination with other neurotrophic factors which act to more particularly enhance a particular differentiation fate of the neuronal progenitor cell. In the later instance, the neuralizing agent might be viewed as ensuring that the treated cell has achieved a particular phenotypic state such that the cell is poised along a certain developmental pathway so as to be properly induced upon contact with a secondary neurotrophic factor. In similar fashion, even relatively undifferentiated stem cells or primature neuroblasts can be maintained in culture and caused to differentiate by treatment with the subject neuralizing agents. Exemplary primative cell cultures comprise cells a harvested from the neural plate or neural tube of an embryo even before much overt differentiation has occurred.
The method of the present invention will also facilitate further determination of a potential role of follistatin as a xe2x80x9cmorphogenxe2x80x9d, that is, a molecule whose tight threshold of concentration determines specific cell fate during development (Wolpert, L. (1969) J. Theor Biol. 25:1-47). One of the first molecules to qualify as a morphogen was bicoid, a DNA binding protein whose graded distribution in the syncytium of the Drosophila embryo leads to the generation of specific cell fates (Driever et al. (1988) Cell 54:95-104). More recently two factors, activin in Xenopus embryos (Green et al. (1992) Cell 71:731-739) and decapentaplegic (dpl), (Ferguson et al. (1992) Cell 71:451-461)) in Drosophila embryos have been showed to act as morphogens in vitro. Both of these factors belong to the TGF-xcex2 superfamily of peptide growth factors and both can specify different cell fates at tight thresholds of concentration. Since follistatin is an inhibitor of activin and both activin ligand and receptor RNAs are expressed in the presumptive ectoderm, follistatin, like activin, may have morphogenic activity. Both the dominant negative activin receptor and follistatin, as described below, elicit neural tissue formation directly. Based on this data, it is asserted that neural tissue represents the default state vis-a-vis activin signaling in presumptive ectoderm. In this model, the amount of activin ligand and receptor present in the cap maintains the cells as epidermal, and additional activin changes the cells"" fate to mesodermal. By inhibiting activin to varying degrees, follistatin could also act as a morphogen.
In an illustrative embodiment of an in vitro assay system, to test if follistatin (or another NA) can act as a morphogen, dissociated animal cap cells can be cultured and dosed with activin at a concentration sufficient to turn on a general mesodermal marker such as brachyury upon reassociation. The activity of activin can then be challenged with small incremental changes in follistatin protein concentration. Indicators that follistatin might serve as a morphogen will include the observation of small concentration differences giving rise to different cell fates, distinguishable by histology or through the use of cell-type specific molecular markers. These studies will allow for the determination of the extent of the number of independent cellular fates that exist in the presumptive ectoderm as well as, what proportion of animal cap cells become neural in response to different concentrations of follistatin.
Similar studies can be performed with stem cells, such as the neural crest cells described above, to determine if the concentration of follistatin (or other NA) is influential on the path of neuronal differentiation of uncommitted and committed progenitor cells, and ultimately whether concentration effects the particular terminally-differentiated derivation of the progenitor cells which arise.
In another embodiment, in vitro cell cultures can be used for the identification, isolation, and study of genes and gene products that are expressed in response to disruption of activin signaling, and therefore likely involved in neurogenesis. These genes would be xe2x80x9cdownstreamxe2x80x9d of the activin signal, and required for neuronal differentiation. For example, if new transcription is required for the neuralization, a subtractive cDNA library prepared with control animal caps and animal caps treated with follistatin can be used to isolate genes that are turned on or turned off by this process. The powerful subtractive library methodology incorporating PCR technology described by Wang and Brown is an example of a methodology useful in conjunction with the present invention to isolate such genes (Wang et al. (1991) Proc.Natl.Acad.Sci. USA 88:11505-11509). For example, this approach has been used successfully to isolate more than sixteen genes involved in tail resorption with and without thyroid hormone treatment in Xenopus. Utilizing control and treated caps, the induced pool can be subtracted from the uninduced pool to isolate genes that are turned on, and then the uninduced pool from the induced pool for genes that are turned off. From this screen, it is expected that two classes of mRNAs can be identified. Class I RNAs would include those RNAs expressed in untreated caps and reduced or eliminated in induced caps, that is the down-regulated population of RNAs. Class II RNAs include RNAs that are upregulated in response to induction and thus more abundant in treated than in untreated caps. RNA extracted from treated vs untreated caps can be used as a primary test for the classification of the clones isolated from the libraries. Clones of each class can be further characterized by sequencing and, their spatiotemporal distribution determined in the embryo by whole mount in situ and developmental northern blots analysis.
For example, in one embodiment of this subtractive assay, special attention can be given to genes that prove to be an immediate early response to neural induction. To qualify as such, these genes should fulfill the following four criteria. First, the RNA should appear quickly (10 to 30 minutes) following application of the inducer. To test this requirement, RNA can be isolated at different times from induced caps and scored for gene expression by northern blots. Second, the induction of the gene should not require previous protein synthesis. Thus, caps can be incubated with cycloheximide (5 xcexcg/ml) prior to and during short incubation with follistatin (30 minutes) after which the caps can be allowed to remain in follistatin for longer periods of time (90 minutes) and then analyzed by northern blotting. This strategy has been used in a similar situation when Mix. 1, a homeobox gene exhibiting an immediate early response to both activin and bFGF was isolated from Xenopus animal caps (Rosa, F. M. (1989) Cell. 57:965-974). These conditions are sufficient to inhibit 75% to 80% of the protein synthesis during the period of induction and to abolish the induction of muscle actin mRNA in response to activin in late explants (Bolce et al. (1993) Developmental Biology 160). Third, immediate early response genes should be expressed as a result of contact with the inducer and not from a secondary cell-cell induction. One method to differentiate between these two responses is to dissociate the cells of the animal cap in Ca/Mg free medium, add follistatin and compare the amount of the induced transcript in dissociated cells versus intact caps. If the levels are comparable in both types of caps, then it may be concluded that cell-cell contact was not required for this induction and it is thus likely a direct response of follistatin treatment. Finally, these genes would be expected to be present and activated in the nervous system during neurogenesis.
Once isolated, the genes regulated by follistatin can be sequenced and their embryonic distribution can be determined by wholemount approaches. If their embryonic expression is in agreement with a possible neurogenic function, they can be tested for neuralizing activity in animal caps and in embryos as described herein for follistatin and other NAs.
Moreover, the present invention provides assays for identifying novel neuralizing agents. For example, an assay can comprise animal cap cells, or equivalent cells thereof, cultured in the presence of a TGF-xcex2-type factor which inhibits neuralization such as activin. A portion of the cells are contacted with a candidate agent, and neuronal differentiation of any of the cells, is scored for by the presence of a neuronal marker, such as NCAM, being expressed by the cells.
Other embodiments of the assay can score simply for the ability of an added agent to inhibit protein-protein interaction between a TGF-xcex2 and its cognate receptor. For instance, in one embodiment, the assay evaluates the ability of a compound to modulate binding between an activin polypeptide and an activin receptor. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by skilled artisan.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as xe2x80x9cprimaryxe2x80x9d screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with receptor proteins. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an activin receptor polypeptide which is ordinarily capable of binding an activin protein. To the mixture of the compound and receptor is then added a composition containing an activin polypeptide. Detection and quantification of receptor/activin complexes provides a means for determining the compound""s efficacy at inhibiting complex formation between the receptor protein and the activin polypeptide. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified activin polypeptide is added to a composition containing the receptor protein, and the formation of receptor/activin complex is quantitated in the absence of the test compound.
Complex formation between the activin polypeptide and an activin receptor may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labelled proteins such as radiolabelled, fluorescently labelled, or enzymatically labelled activin polypeptides, by immunoassay, or by chromatographic detection.
Accordingly, a wide range of agents can be tested, such as proteins and polypeptides, as well as peptidomimetics and other small molecules (including natural products). For instance, a drug screening assay described above can be used in the reduction of the activin of inhibin proteins to generate mimetics, e.g. peptide or non-peptide agents, which are able to disrupt binding of an activin polypeptide of the present invention with an activin receptor. By employing, for example, scanning mutagenesis to map the critical amino acid residues of the activin protein involved in binding the activin receptor, peptidomimetic compounds can be generated which mimic those residues in binding to the receptor and which consequently can inhibit binding of activin to its receptor, as may be detected in a screening assay as described herein. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), xcex2-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and xcex2-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71.
In addition to the implantation of cells cultured in the presence of an NA and other in vitro uses described above, yet another objective of the present invention concerns the therapeutic application of activin-disrupting agents to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system. The ability of follistatin to regulate neuronal differentiation not only during development of the nervous system but also presumably in the adult state indicates that NAs can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and death which result from loss of differentiation in certain pathological conditions. In light of this understanding, the present invention specifically contemplates applications of the subject method to the treatment of (prevention and/or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous system including Alzheimer""s disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson""s disease, Huntington""s chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis; and (v) degenerative diseases of the retina.
Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treated with a therapeutic regimen which includes a neuralizing agent of the present invention. For example, Alzheimer""s disease is associated with deficits in several neurotransmitter systems, both those that project to the neocortex and those that reside with the cortex. For instance, the nucleus basalis in patients with Alzheimer""s disease were observed to have a profound (75%) loss of neurons compared to age-matched controls. Although Alzheimer""s disease is by far the most common form of dementia, several other disorders can produce dementia. Many are age-related, occurring in far greater incidence in older people than in younger. Several of there are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalmus or the white matter underlying the cerebral cortex. Here, the cognitive dysfumction results from the isolation of cortical areas by the degeneration of efferents and afferents. Huntington""s disease involves the degeneration of intrastraital and cortical cholinergic neurons and GABAergic neurons. Pick""s disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum. Treatment of patients suffering from such degenerative conditions can include the application of neuralizing agent polypeptides, or agents which mimic their effects, in order to manipulate, for example, the de-differentiation and apoptosis of neurons which give rise to loss of neurons. In preferred embodiments, a source of a neuralizing agent is stereotactically provided within or proximate the area of degeneration.
In addition to degenerative-induced dementias, a pharmaceutical preparation of a neuralizing agent can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements. Parkinson""s disease, for example, primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident. Also included are neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders. Examples include chronic atrophies such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive bulbar palsies or spinal muscular atrophies. The present method is amanable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion. For instance, a preparation of a neuralizing agent of the present invention can be used to treat a restricted form of cerebellar cortical degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.
In yet another embodiment, the subject method is used to treat amyotrophic lateral sclerosis. ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex. The therapeutic application of an activin antagonist can be used alone or in conjunction with other neurotrophic factors such as CNTF, BDNF, or NGF to prevent and/or reverse motor neuron degeneration in ALS patients.
The neuralizing agents of the present invention can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle endocrine tissue (such as glandular tissue). For instance, neuralizing agent compositions may be useful to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.
In yet another embodiment, the subject neuralizing agents can be used in the treatment of neoplastic or hyperplastic transformations, involving neural tissue. For instance, an activin antagonist likely to be capable of inducing differentiation of transformed neuronal cells to become post-mitotic or possibly apoptotic. Inhibition of activin-mediated inductive events may also involve disruption of autocrine loops, such as PDGF autostimulatory loops, believed to be involved in the neoplastic transformation of several neuronal tumors. The subject method may, therefore, be of use in the treatment of, for example, malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.
The NA, or a pharmaceutically acceptable salt thereof, may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, xe2x80x9cbiologically acceptable mediumxe2x80x9d includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the NA, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington""s Pharmaceutical Sciences (Remington""s Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable xe2x80x9cdeposit formulationsxe2x80x9d. Based on the above, the pharmaceutical formulation includes, although not exclusively, NA solutions or a freeze-dried powder of an NA (such as a follistatin) in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. For illustrative purposes only and without being limited by the same, possible composition of formulations which may be prepared in the form of solutions for the treatment of nervous system disorders with an NA are given in the della Valle U.S. Pat. No. 5,218,094. In the case of freeze-dried preparations, supporting excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH. Similar solutions may also be used for the pharmaceutical compositions of the NA in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, for example, neutral pH.
Methods of introduction of the NA at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Methods of introduction may also be provided by rechargable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an NA at a particular target site. Such embodiments of the present invention can be used for the delivery of an exogenously purified NA, which has been incorporated in the polymeric device, or for the delivery of an NA produced by a cell encapsulated in the polymeric device.
An essential feature of certain embodiments of the implant is the linear release of the NA, which can be achieved through the manipulation of the polmer composition and form. By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled. The selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical and Dental Materials, ed. by David Williams (MIT Press: Cambridge, Mass., 1990); and the Sabel et al. U.S. Pat. No. 4,883,666. In another embodiment of an implant, a source of cells producing the NA, or a solution of hydrogel matrix containing purified NA, is encapsulated in implantable hollow fibers. Such fibers can be pre-spun and subsequently loaded with the NA source (Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can be co-extruded with a polymer which acts to form a polymeric coat about the NA source (Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat. No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55).
In yet another embodiment of the present invention, the neuralizing agent can be administered as part of a combinatorial therapy with other agents. For example, the combinatorial therapy can include an neuralizing agent such as follistatin with at least one trophic factor. Exemplary trophic factors include nerve growth factor, ciliary neurotrophic growth factor, schwannoma-derived growth factor, glial growth factor, strectal derived neuronotrophic factor, platelet-derived growth factor, and scatter factor (HGF-SF). Other neural inductive proteins, such as hedgehog-like proteins, noggin, and ligands of the Notch receptor, may also be used in conjunction with the subject neuralizing agent. Antimitogenic agents can also be used, as for example, when proliferation of surroundig glial cells or astrocytes is undesirable in the regeneration of nerve cells. Examples of such antimitotic agents include cytosine, arabinoside, 5-fluorouracil, hydrozyurea, and methotrexate.
Moreover, certain of the neuralizing agents, such as the dominant negative activin receptors (either soluble of membrane bound), may be ammenable to delivery by gene therapy. For instance, expression constructs of the subject dominant negative receptors may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the dominant negative receptor gene to cells in vivo. Approaches include insertion of the mutant receptor gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. While viral vectors transfect cells directly, plasmid DNA can also be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically. Furthermore, it will be recognized that the particular gene construct provided for in vivo transduction of activin expression are also useful for in vitro transduction of cells, such as for use in the ex vivo tissue culture systems described above.
The fact that neuralization was closely linked to mesoderm induction has hampered most of the previous effort invested in the molecular characterization of neural inducers and patterners. Thus, attempts to isolate factors involved in neural induction and patterning have ended in the identification and characterization of mesoderm inducers and modifiers. The data described in the Examples below demonstrate that two activin antagonists, the dominant negative form of the activin receptor (xcex941XAR1) and follistatin both elicit direct neuralization in embryonic explants without a prerequirement for mesoderm induction. In addition, a full length cDNA for Xenopus follistatin has been isolated and its embryonic localization shown to be in perfect agreement with a role in neural development in vivo. The observation that antagonizing the activin signal results in neuralization suggests, for the first time, that neural induction in vertebrates represents a default state.
Additionally, as described in the Examples below, the truncated activin receptor does not block mesoderm induction by exogenous FGF in animal caps, and yet endogenous FGF does not induce mesoderm in a significant fraction of embryos injected with xcex941XAR1. Furthermore, when half the embryo is injected with xcex941XAR1, that half lacks Xbra expression in all embryos tested (n=25) even though FGF is a potent inducer of brachyury in the animal cap assay. These data indicates that endogenous FGF signaling is not sufficient to rescue brachyury expression or mesoderm induction in the marginal zone of embryos injected with xcex941XAR1. At the same time, it is clear from other experiments with a dominant negative FGF receptor that FGF plays an important role in axial patterning, particularly for posterior structures (Amaya et al. (1991) Cell. 66:257-270). Taken together, these findings raise the possibility that FGF signaling at the time of mesoderm induction requires a functional activin pathway.